The human -type globin genes are expressed exclusively in erythroid cells and regulated by a locus control region (LCR) that is located far upstream of the genes. Mutations in the -globin locus are among the most common disease-causing mutations in the human population. These mutations lead to thalassemias, characterized by reduced or absent adult -globin gene expression, or sickle cell anemia. A possible therapy for these hemoglobinopathies is the reactivation of the normally repressed -globin genes in adult erythroid cells. We propose to continue our efforts to understand how the LCR regulates globin gene expression during development and differentiation and will develop new methodology for changing expression patterns in the - globin gene locus. We established a novel procedure utilizing small synthetic DNA binding domains to analyze and neutralize specific cis-regulatory DNA elements in the -globin gene locus. We will target these synthetic DNA binding domains to known repressor binding sites in the -globin gene promoters and to activator binding sites in the gene encoding for the -globin repressor Bcl11A. Targeting these sites with synthetic DNA binding proteins is expected to increase -globin expression in adult erythroid cells. We and others have shown previously that the LCR recruits transcription complexes which produce enhancer RNAs (eRNAs). Transcription factors NF-E2 and USF have been implicated in transcription complex recruitment to the LCR. We will utilize the synthetic DNA binding domains to analyze the contribution of cis-regulatory elements in the LCR that interact with transcription factors NF-E2 and USF. Furthermore, we will examine if LCR associated transcripts or the process of transcription contributes to LCR mediated activation of globin gene expression. The four aims of this proposal will focus on optimizing the DNA binding specificity and delivery methods for synthetic DNA binding proteins (aim 1), to use synthetic DNA binding proteins to analyze cis-regulatory elements in the -globin locus and to reactivate -globin expression in adult erythroid cells (aim 2), to identify all proteins associated with the human LCR in erythroid cells of transgenic mice (aim 3), and to analyze the mechanism(s) by which the LCR and LCR associated non-coding transcription activates the globin genes (aim 4).